Extracts from hop, methods for producing the same and their use

ABSTRACT

Novel extracts from hop with an increased content of prenylated chalcones and flavones, methods for producing the same, pharmaceutical preparations comprising such extracts of hop and use of these extracts of hop for the prophylaxis and treatment of pathological conditions caused by oestrogen deficiency or by dysregulations to sex-hormone-related metabolism are described.

The present invention relates to extracts from hop, methods forproducing the same and their use for the prophylaxis and treatment ofpathological conditions caused by oestrogen deficiency or bydysregulations to sex-hormone-related metabolism, in particularoestrogen metabolism.

The greatest importance of hop is still its use for producing beer. Dueto its bittering and flavouring agents it is decisive for the taste ofbeer. Moreover, these agents have reached a certain relevance inconservating beer because of its antimicrobiotic characteristics.

The scientific knowledge in the field of hop in the early 80's resultedin a positive monograph from the agency E of the federal healthauthorities at that time (Bundesanzeiger dated Dec. 5, 1985 and Mar. 13,1990, respectively). Thus, use of hop for the treatment of sleepdisturbances, agitation and anxiety is basically permitted.

Already for a long time hop has a pharmaceutical relevance as a mildsedative in public medicine. Presumably α- and β-bitter acids which aresensitive to oxidations are responsible for this effect. In recent timesit was shown that these ingredients also exhibit radical trapcharacteristics and lipid peroxidation inhibiting properties (M.Tagashira et al., Biosci. Biotech. Biochem. 59, 740-742 (1995)).Furthermore, pharmaceutical compositions for the treatment ofosteoporosis are described in the European patent application 0 677 289A2 which contain compounds from the group of α-bitter acids andα-iso-bitter acids.

In the last years also the phenolic ingredients of hop were increasinglyexamined beside α- and β-bitter acids (J. H{haeck over (o)}lzl,Zeitschrift für Phytotherapie 13, 155-161 (1992)) and beside the longerknown Xanthohumol 1 further flavone-type compounds were found in hopplants (J. F. Stevens et al., Phytochemistry 44, 1575-1585 (1997), J. F.Stevens et al., J. Chromat. A 832 (1-2), 97-107 (1999)). These wereprimarily isoprenylated flavonoides, such as for example 6- or8-prenylnaringenine 2 and 3 and isoxanthohumol 4. Stevens et al.(Phytochemistry 53, 759-775 (2000)) also examined the chemotaxonomy ofspecies of hop and taxa of hop.

Again and again it was observed that menstrual disorders appeared inwomen that picked hop, which were traced back to oestrogenic substancesin hop, but these effects could not clearly be associated with one ormore ingredients. In the meantime this oestrogenic activity of hop couldbe confirmed. Thus, it was demonstrated that 8-prenylnaringenine 3 isessentially responsible for these effects (S. R. Milligan et al., J.Clin. Endocrinol. Metab. 84, 2249-2252 (1999)). The oestrogenic activityin vitro of this compound appeared from its relative binding affinity tooestrogenic receptors and was particularly tested by stimulatingalkaline phosphatase in Ishikawa-Var-I-cells. Thus, it was demonstratedthat 8-prenylnaringenine was significantly more active than thephytooestrogenes known so far such as coumestrol, genistein or daidzein,and produced only a slightly weaker effect than 17β oestradiol. Milliganet al. (J. Endocrin. Metabol. 85, 4912-4915 (2000)) also reported aboutthe bonding of different phenolic ingredients of hop to a humanoestrogenic receptor expressed in yeast cells. Thereby, again8-prenylnaringenine exhibited the most powerful oestrogenic activity.6-Prenylnaringenine, 6,8-diprenylnaringenine and 8-geranylnaringenineexhibited weaker oestrogenic properties. Miyamoto et al. (Planta Med.64, 516-519 (1998)) could demonstrate that 8-prenylnaringeninenormalises the weight of the uterus and osseous density atovariectomised rats. Moreover, the oestrogen agonistic activity of aseries of 8-prenylated flavone derivatives including 8-prenylnaringenineis described in JP 08 165238 (ref. CA 125:158632).

Recent studies could demonstrate that some flavonoides of hop,particularly xanthohumol 1, can influence the cell metabolism. They arecapable of positively influence enzymatic reactions that play animportant role in the development of tumour cells. For this reason thesecompounds can be considered as preventive agents against cancer(conference of the German Society of Hop Research, present GermanSociety of Hop Research, present knowledge concerning the hop ingredientxanthohumol, Mar. 24, 1998, Aschheim). Miranda et al. (Food Chem. Tox.37(4), 271-285 (1999)) reported strong antiproliferative activity ofxanthohumol 1 and isoxanthohumol 4 in human breast cancer cells MCF-7,colon cancer cell lines HAT-29 and ovarian cancer cell lines A-2780.

It could further be demonstrated that xanthohumol 1 has an inhibitingeffect on osteolysis. Its use as a therapeutic agent againstosteoporosis is described in the European patent EP 0 679 393 B1.Although the inventors postulate oestrogenic properties of xanthohumol,these properties are not demonstrated. Contrarily, S. R. Milligan et al.(Pharm. Pharmacol. Lett. 7, 83-86 (1997)) clearly excluded that theosteoporosis inhibiting activity of xanthohumol is based on anoestrogenic effect because the corresponding activities could not bedetected for both the human endometrial carcinoma cell line Ishikawa andin an yeast reporter gene assay (S. R. Milligan et al., J. Clin.Endocrinol. Metabol. 84, 2249-2252 (1999)). Contrarily to theseexaminations the present invention demonstrates that xanthohumol 1 andisoxanthohumol 4 having a comparable activity bind to the oestrogenicreceptors alpha and beta.

Kumai and Okamoto (Toxicology Letters 21, 203-207 (1984)) reported abouthigh-molecular carbohydrate fractions from merely aqueous extracts ofhop reducing the ovarian weight of young rats pretreated with PMSgonadotropin. Okamoto and Kumai (Acta Endocrinologica 127, 371-377(1992)) confirmed these results on the basis of the observation ofreduced blood levels of 17β oestradiol and LH caused by administeringthe merely aqueous extract from hop.

An extract from hop having an increased content of xanthohumol isdescribed in the German patent application DE 199 39 350 A1. Thisextract is to be added to beer and to soft drinks containing fruitjuice. There is nothing known about the presence of prenylatednaringenines in this extract. According to an embodiment xanthohumol isextracted from the hop with 50% by weight ethanol. However, this doesnot result in an ideal extraction of xanthohumol because a high degreeof transition into the extract cannot be obtained, until ethanol havinga high content (>80% by weight) is used.

A method for the isolation of oestrogenically active compounds from hopis claimed in WO 83/00701 A1, characterised in that a carbon dioxideextract from hop is produced first adding water as an entraining agentand subsequently the oestrogenically active compounds are obtainedtherefrom by means of an extraction using ether or chromatographicmethods. Moreover, the use of these compounds as an additive for animalfeed, for cosmetic agents or as a bathing additive is claimed. There donot appear any particulars about the type of these oestrogenicallyactive compounds.

A method for the isolation of stable additives for brewing beer isclaimed in WO 01/30961 A1, characterised in that the residue of the hopdraff from the carbon dioxide extraction is extracted with a polarsolvent, preferably hot water and the extract is subsequently acidified,washed with a nonpolar solvent, preferably hexane, and—optionally afterdrying—used as a brewing additive. The remaining draff is discarded.

The object underlying the present invention is to provide plant extractsthat are suitable for the production of pharmaceuticals for theprophylaxis and treatment of pathological conditions that are caused byan oestrogen deficiency or by dysregulations to sex-related-homonemetabolism, in particular oestrogen metabolism.

A further object of the present invention is to provide a method forproducing such extracts and pharmaceutical preparations comprising thesame suitable for the treatment of the pathological conditions mentionedabove.

According to the present invention these objects are solved by theextract from hop according to claims 1 and 2, the methods according toclaims 3 to 12, the pharmaceutical preparation according to claim 13 andthe use of the extracts or the pharmaceutical preparation according toclaims 14 to 16.

The present invention relies inter alia on the surprising observationthat after removing lipophilic and hydrophilic fibres extracts areobtained from the hop drug still containing phloroglucinol-type bitteracids of hop and simultaneously containing free and/or bonded chalconesand flavones such as xanthohumol, isoxanthohumol as well as 6- and8-prenylnaringenine in an enriched form. The fact that the content of 6-and 8-prenylnaringenine depends on the temperature of the preextractionwith water (cf. Example 3) and that it can be enhanced by a factor of upto about 2 is particularly surprising.

FIG. 1 demonstrates the dependence of the concentration of the analysedingredients on the temperature of the preextraction with water.

Such an extract can be obtained by one or more extractions using aC₅-C₇-alkane or supercritical CO₂ (step of removing fat), subsequentextraction of the remaining drug residue using water and followed byextraction of the still remaining drug residue using a solvent of mediumpolarity selected from the group consisting of alcohols, aqueousalcohols, ketones, aqueous ketones, esters and optionally subsequentliquid-liquid distribution. Surprisingly the transition of the bitteracids of hop into the lipophilic extract does not occur completely butonly in parts. On the other hand, the chalcones and flavones remainnearly completely in the drug residue during the extraction using water.Thereby, an extract from hop is obtainable containing all of thepharmacologically relevant ingredients (bitter acids, chalcones,flavones) in a balanced ratio. Due to this advantageous compositionusing several therapeutic principles this extract is ideally applicableagainst pathological conditions caused by oestrogen deficiency or byother hormone-related dysregulations.

The extracts from hop according to the present invention are suitablefor the prophylaxis and treatment of climacteric-related complaints orpost menopause-related complaints in women, the symptoms comprisinginter alia hot flushes, depression, anxiety, mental confusion, insomniaand post menopause-related serious health problems such as osteoporosis,diseases of the cardiovascular system, cerebral infarction (strokes),dementia and tumour diseases. Other diseases which are based on adysregulation of the sex-hormone-related metabolism and which can betreated using the extract according to the present invention are forexample amenorrhea, anovulatory cycles, menometrorrhagia, premenstrualcomplaints and postpartal depressions. Likewise these extracts can beused for the treatment of sex-hormone-dependent diseases in men such asfor example benign prostatic hypertrophy or carcinoma of the prostate.

The surprisingly high oestrogenic activity of the extracts from hopaccording to the present invention were detected both using acompetitive receptor binding assay for the human oestrogen receptorsalpha and beta and in a recombinant yeast assay compared to the activityof 17β oestradiol. Contrarily, conventional standard extracts of hophaving the same dosage exhibit an essentially lower activity or noactivity at all. FIG. 2 illustrates the activity of the comparativeextract and of two extracts of hop according to the present invention ina yeast reporter gene assay.

According to the present invention, an extract from hop having anincreased content of free and/or bonded chalcones and flavones, inparticular 6- and 8-prenylnaringenine, xanthohumol and isoxanthohumolcompared to conventional, in particular aqueous alcoholic extracts isprovided, which simultaneously further contains α and possibly β bitteracids (humulone and lupolone and derivatives thereof, respectively).

Furthermore, according to the present invention a method for thepreparation of these extracts from hop is provided, comprising the stepsof:

-   (a) one or more extractions of a hop drug using a C₅-C₇-alkane or    supercritical CO₂ and separating the drug residue from the    extraction solution;-   (b) one or more extractions of the drug residue obtained in step (a)    using water and separating the drug residue;-   (c) one or more extractions of the drug residue obtained in step (b)    using a solvent selected from the group consisting of alcohols,    aqueous alcohols, ketones, aqueous ketones and esters and filtration    of the extraction solution obtained; and-   (d) removing the solvent from the combined extraction solutions    obtained in step (c) and drying of the residue obtained.

The drug-to-solvent ratio for every extraction step is in the range ofabout 1:7 to about 1:12.

The extraction using a C₅-C₇-alkane or supercritical CO₂ in step (a) ispreferably carried out once, twice or three times, particularly threetimes.

The extraction using supercritical CO₂ is particularly preferred.

The C₅-C₇-alkane used in step (a) is preferably a C₅-C₇-n-alkaneselected from the group consisting of n-pentane, n-hexane, andn-heptane, with n-heptane being particularly preferred.

The extraction according to step (b) is preferably carried out at atemperature between 60 and 95° C., preferably at 90° C., wherein theduration of the extraction can take one or more hours.

The solvent used in step (c) is preferably selected from the groupconsisting of ethanol, aqueous ethanol, methanol, aqueous methanol,acetone, aqueous acetone and ethyl acetate, with 80 to 96% (w/w)ethanol, 74 to 99% (w/w) methanol and 60 to 99% (w/w) acetone beingpreferred, respectively, and 92% (w/w) ethanol being particularlypreferred.

The (dry) extract from hop according to the present invention ischaracterised by having a content of a bitter acids of at least 0.5%,preferably at least 0.8% and particularly preferred 1%, a content ofxanthohumol of at least 2%, preferably at least 3% and particularlypreferred 4%, and a content of prenylated flavones of at least 0.5%,preferably at least 0.7%. The prenylated flavones preferably comprise6-prenylnaringenin, 8-prenylnaringenin and isoxanthohumol. For thepurpose of the present invention xanthohumol is not among the prenylatedflavones. Percentage details refer to the weight of the dry extract fromhop.

The extracts obtained can be processed together with conventionalpharmaceutically acceptable additives to pharmaceutical preparationssuch as capsules, film tablets or coated tablets. Fillings, bondingagents, disintegrants, lubricants and coatings for film tablets andcoated tablets, as well as oils and fats as filling agents for gelatinecapsules, can be used as pharmaceutical additives.

The extracts according to the present invention can be used for theprophylaxis and treatment of pathological conditions, caused byoestrogen deficiency or by other hormone-related dysregulations, such asparticularly climacteric complaints, sex-hormone-dependent cancerdiseases, benign prostatic hypertrophy, osteoporosis, Alzheimer'sdisease and diseases of the cardiovascular system. In case ofsex-hormone-dependent cancer diseases the extracts according to thepresent invention can be particularly used for the prophylaxis and thetreatment of breast cancer, carcinoma of the uterus and prostaticcarcinoma.

The dosage of the extracts according to the present invention is in therange from 0.005 g to 2 g extract 1 to 4 times per day, preferably inthe range from 0.02 g to 1 g 1 to 2 times per day. In an individualcase, the dosage depends on the clinical picture and on the individualcircumstances of the patient and can be adjusted to the respectiverequirements by the attending practitioner.

The examples given below illustrate the invention and should not beconsidered to limit the invention. All percentage details refer to theweight, unless specified otherwise.

COMPARATIVE EXAMPLE Production of an Extract Using 96% (w/w) EthanolWithout Prior Degreasing

50 g of drug of hop (sort “Hallertauer Magnum”) were mixed with 500 g96% (w/w) ethanol and reduced to small peaces using an ultraturrax. Theextraction was carried out for 1 hour at 60° C. Then the extract wasfiltered using a filter type Seitz 1500. The drug was extracted further2 times in the same way. The combined extraction solutions were set freefrom the ethanol on a rotary evaporator and dried overnight at 50° C. ina vacuum drying cabinet. The content of characteristic ingredients wasdetermined from the dry matter using the following HPLC method. ThisHPLC method is also applied for the determination of ingredients in theother examples. Column LiChrospher 100 5 μm. 250 × 4 mm Eluant A: 1000ml bidest. water/3 ml phosphoric acid (85%)/2 ml triethylamine B: 1000ml acetonitrile/3 ml phosphoric acid (85%)/2 ml triethylamine/60 mlbidest water Gradient from 40% B to 70% B within 30 min; from 70% B to100% B within 10 min Flow 1.2 ml/min Detection diode array Yield (96%(w/w) 18.38 g => 36.8% ethanol extract): HPLC content of 19.8% α bitteracid of hop: HPLC content of  4.2% β bitter acid of hop: HPLC content 1.3% of xanthohumol: HPLC content of below 6- and 8-prenylnaringeninesthe detection as well as limit (<0.01%) isoxanthohumol:

Example 1a Production of an Extract from Hop (Extraction Using CO₂ andSubsequent Preextraction at 90° C. Using Water)

Sequential extraction using supercritical CO₂, water and 92% (w/w)ethanol:

80.6 g of a drug of hop (sort “Hallertauer Magnum”) which had beenpreextracted using supercritical CO₂ (conditions: milling to a grainsize of 10 mm, extraction using CO₂ at 250 bar/50° C., separating theextract with a yield of 30%) were initially extracted using 960 g ofwater for 5 minutes on an ultraturrax, then extracted for 1 hour at 90°C. while being stirred. Subsequently the water extract was filtered overa Seitz Supra Filter 1500. Then the still somewhat wet drug residue wasextracted twice using 800 g of 92% (w/w) ethanol for 1 hour at 60° C.,respectively. Subsequently it was filtered over a Seitz Supra 1500 andthe extraction solution was set free from ethanol on a rotary evaporatorhaving a temperature of the water bath of 55 to 65° C. and dried at 60°C. in the drying cabinet. Yields: Residue from water extraction: 18.96 g(23.5%) Residue from the extraction using 92% (w/w) EtOH:  9.83 g(12.2%) HPLC contents (based on the extract using 92% (w/w) EtOH): HPLCcontents of α bitter acids of hop:   2% HPLC contents of β bitter acidsof hop:  0.5% HPLC content of xanthohumol 5.83% HPLC content of6-prenylnaringenine 0.63% HPLC content of 8-prenylnaringenine 0.21% HPLCcontent of isoxanthohumol 0.42%

Example 1b Production of an Extract from Hop (Extraction Using CO₂ andSubsequent Preextraction at 90° C. Using Water)

Sequential extraction using supercritical CO₂, water and 92% (w/w)ethanol:

504.26 g of a drug of hop (sort “Hallertauer Magnum”) which had beeninitially preextracted using supercritical CO₂ (conditions: milling to agrain size of 10 mm, extraction using CO₂ at 250 bar/50° C., separatingthe extract with a yield of 30%) were extracted using 6 kg of waterinitially for 5 minutes on an ultraturrax, then extracted for 1 hour at90° C. while being stirred. Then the water extract was filtered over aSeitz Supra Filter 1500. Then the still somewhat wet drug residue wasextracted twice using 5 kg of 92% (w/w) ethanol for 1 hour at 60° C.,respectively. It was filtered over a Seitz Supra 1500 and the extractionsolution was set free from ethanol on a rotary evaporator having atemperature of the water bath of 55 to 65° C. and dried at 60° C. in adrying cabinet. Yields: Residue from the extraction using water: 105.9 g(21%) Residue from the extraction using 92% (w/w) EtOH: 69.37 g (13.8%)HPLC contents (based on the extract using 92% (w/w) EtOH): HPLC contentof α bitter acids of hop:   1% HPLC content of β bitter acids of hop: 0.5% HPLC content of xanthohumol 4.41% HPLC content of6-prenylnaringenine 0.49% HPLC content of 8-prenylnaringenine 0.15% HPLCcontent of isoxanthohumol  0.6%

Example 2 Production of an Extract from Hop (Extraction Using CO₂ andSubseqdently Preextraction at 60° C. Using Water)

Sequential extraction using supercritical CO₂, water and 92% (w/w)ethanol:

80.36 g of a drug of hop (sort “Hallertauer Magnum”) which had beenpreextracted using supercritical CO₂ (conditions: milling to a grainsize of 10 mm, extraction using CO₂ at 250 bar/50° C., separating theextract with a yield of 30%) were initially extracted using 964 g ofwater for 5 minutes on an ultraturrax, then extracted for 1 hour at 60°C. while being stirred. Then the water extract was filtered over a SeitzSupra Filter 1500. Then the still somewhat wet drug residue wasextracted twice using 800 g of 92% (w/w) ethanol initially for 5 minuteson an ultraturrax, respectively, subsequently extracted for 1 hour at60° C. while being stirred, respectively. Then it was filtered over aSeitz Supra 1500 and the extraction solution was set free from ethanolon a rotary evaporator having a temperature of the water bath of 55 to65° C. and dried at 60° C. in the drying cabinet. Yields: Residue fromthe extraction using water: 17.91 g (22%) Residue from the extractionusing 92% (w/w) EtOH:  9.95 g (12.4%) HPLC contents (based on theextract using 92% (w/w) EtOH): HPLC content of α bitter acids of hop:1.58% HPLC content of β bitter acids of hop:   0% HPLC content ofxanthohumol  6.1% HPLC content of 6-prenylnaringenine  0.4% HPLC contentof 8-prenylnaringenine 0.09% HPLC content of isoxanthohumol 0.21%

Example 3 Production of an Extract from Hop (Extraction Using n-Heptaneand Subsequently Water at 90° C.)

247.6 g of a drug of hop (sort “Hallertauer Magnum”) were extractedusing 7 times its weight made up of n-heptane initially for 5 minutes onan ultraturrax, then extracted for 1 hour while being stirred. Afterfiltering the heptane extraction solution over a Seitz Supra 1500 it wasextracted for a second time in the same way. Afterwards the obtaineddrug residue was set free from heptane in a vacuum drying cabinet. Thedry drug residue (205 g) was then mixed with 12 times its weight made upof water and maintained for 1 hour at 90° C. Subsequently it wasfiltered again and the still somewhat wet drug residue was extractedtwice at 60° C. using 10 times its weight made up of 92% (w/w) ethanolwhile being stirred. It was then filtered over a Seitz Supra 1500 andthe extraction solution was set free from ethanol on a rotary evaporatorhaving a temperature of the water bath of 55 to 65° C. and dried at 60°C. in a drying cabinet. Yields: extract using heptane: 26.4 g (10.7%)extract using water: 41.1 g (16.6%) extract using 92% (w/w) ethanol:52.0 g (21.0%) HPLC contents (based on the extract using 92% (w/w)ethanol): α bitter acids: 0.86% β bitter acids: 0.05% xanthohumol:  3.3%6-prenylnaringenine 0.45% 8-prenylnaringenine 0.13% isoxanthohumol 0.25%

Example 4 Dependence of the Content of 6-Prenylnaringenin,8-Prenylnaringenin, and Isoxanthohumol on the Temperature of thePreextraction Using Water

Extraction: About 80 g of a drug of hop being preextracled using CO₂were extracted using 12 times its weight made up of water initially for5 minutes on a ultraturrax, then extracted for 1 hour at 60, 70, 80, 90,and 95° C. while being stirred. Then the extract using water wasfiltered over a Seitz Supra Filter 1500. Subsequently the still somewhatwet drug residue was extracted twice using 800 g of 92% (w/w) ethanolinitially for 5 minutes on an ultraturrax, respectively, then extractedfor 1 hour at 60° C. while being stirred. It was then filtered over aSeitz Supra 1500 and the extraction solution was set free from ethanolon a rotary evaporator having a temperature of the bath of 55 to 65° anddried at 60° C. in a drying cabinet.

The results being graphically represented in FIG. 1 exhibit asignificant dependence of the concentration of the analysed prenylatedingredients on the temperature of the preextraction using water.

Example 5 Testing the Extracts from Hop for Oestrogenic Activity

The testing of individual ingredients of extracts, a comparative extractand an extract according to the present invention for interactions withthe human oestrogenic receptor alpha (ER-α) and beta (ER-β),respectively, a competitive receptor binding assay was carried out.Thereby, a radioactively labelled oestradiol was initially bonded to thehuman oestrogen receptor and then treated with the test substance to beexamined. A portion of the labelled oestradiol which corresponds to theoestrogenic potency of the sample is thus displaced. Excessiveoestradiol is washed out after bonding of the complex tohydroxylapatite. The oestrogenic receptors ER-α and ER-β werecommercially available as recombinant human receptors. The testpreparations consisted of 1000 μl TEDG buffer (10 mM tris, 1,5 mM EDTA,10% glycerol, pH 7.5), 5 μl receptor (200 nM), 10 μl 3H-oestradiol and10 μl ethanol (control value), 10 μl diethylstiloestrol (100 mM,positive control) or 10 μl extract or ingredient of the extract,respectively. The preparations were carefully mixed and incubated forabout 16 hours at room temperature in the dark. After incubation 250 μlhydroxylapatite (HAP) were added to absorb the proteins. During a periodof incubation of 15 minutes the preparations were manually mixed with atime-lag of 5 minutes, respectively. The precipitate is separated bycentrifugation at 10,000 rpm for a few seconds and the supernatantliquid is separated by pipetting. The pellet is washed three times with1000 μl TEDG buffer, respectively, 1000 μl ethanol are added for themeasurement, the mixture is slurried and transferred to a scintillationvial. After adding 9 ml scintillator liquid (Ready Safe, Beckmann) ameasurement over the complete ³H window is carried out using a BeckmannBeta-Counter.

The characterisation of the bonding capacities of the test substancesresults from the determination of the ED₅₀ values taken from thedosis-effect-plots of the displacement of oestradiol. The results arereported in Table 1 and demonstrate potent interactions with bothoestrogen receptors for all ingredients examined. Surprisingly theextract according to the present invention was proved to be essentiallymore effective than it had to be expected due the activities of thesingle ingredients. In contrast, the comparative extract exhibited anactivity to both receptors which was at least 10 times smaller than thatof the extract according to the present invention.

Table 1: Bonding of ingredients of the extracts, an extract according tothe present invention and a comparative extract to the human oestrogenreceptor-alpha (ER-α) and oestrogen receptor-beta (ER-β), respectively.TABLE 1 Bonding of ingredients of the extracts, an extract according tothe present invention and a comparative extract to the human oestrogenreceptor-alpha (ER-α) and oestrogen recector-beta (ER-β), respectively.ED₅₀ [pg/ml] relative potency Substance ER-α ER-β3 ER-α ER-β$\frac{{relative}\quad{potency}\quad{ER}\text{-}\alpha}{{relative}\quad{potency}\quad{ER}\text{-}\beta}$17β-Oestradiol 507 400 1 1 1 8-Prenylnaringenine 4.6 × 10⁴ 1.0 × 10⁵ 1.1× 10⁻² 4.0 × 10⁻³ 2.72 6-Prenylnaringenine 1.6 × 10⁶ 4.6 × 10⁵ 3.2 ×10⁻⁴ 8.7 × 10⁻⁴ 0.37 Isoxanthohumol 2.0 × 10⁶ 8.5 × 10⁵ 2.5 × 10⁻⁴ 4.7 ×10⁻⁴ 0.54 Xanthohumol 2.0 × 10⁶ 1.2 × 10⁶ 2.5 × 10⁻⁴ 3.3 × 10⁻⁴ 0.78Extract according to 3.9 × 10⁵ 2.7 × 10⁵ 1.3 × 10⁻³ 1.5 × 10⁻³ 0.87 thepresent invention Comparative extract 4.0 × 10⁶ 4.3 × 10⁶ 1.3 × 10⁻⁴ 9.4× 10⁻⁵ 1.33

Moreover, testing of extracts for oestrogenic properties was carried outwith a reporter gene assay using yeast cells (saccharomyces). The cellsare stably transfected with the human α-oestrogen receptor and anexpression plasmid containing an oestrogen response element and the genefor the enzyme β-galactosidase. All samples were dissolved in DMSO at aconcentration of 20 mg/ml, and were given undiluted or after dilutingwith DMSO at a ratio of 1/10, 1/100 or 1/1000 at a volume of 1 μl to 100μl culture medium in 96-well flat-bottom micro-titre dishes. Next, 100μl yeast suspension and the chromogenic substrate chlorophenolred-β-D-galactopyranoside were added. Control wells were provided onevery dish, which were filled with either the culture medium or thesolvent alone, or which contain the standard concentration of 17βoestradiol. The yeast cells were incubated for 72 hours at 32° C., afterwhich absorption of the medium was measured at 540 nm in a micro-titredish photometer. The samples were partially checked twice.

Results: Sample activity Extract using 96% (w/w) ethanol inactiveaccording to comparative example Extract using 92% (w/w) ethanol activeaccording to Example 1a Extract using 92% (w/w) ethanol active accordingto Example 2

The results of the assays are depicted in FIG. 2. Hereby, those extractsare categorized as “active”, whose activity is significantly above thebackground values (corresponding to about 10% of the maximumstimulation) compared to the 17β oestradiol standard plot.

1. Method for obtaining an extract from hop, comprising the steps of: (a) one or more extractions of a drug from hop with a C₅-C₇-alkane or supercritical CO₂ and separating the drug residue from the solution; (b) one or more extractions of the drug residue obtained in step (a) with water at a temperature in the range of 60 to 95% and separating the drug residue; (c) one or more extractions of the drug residue obtained in step (b) with 80-96% (w/w) ethanol and filtration of the obtained extraction solution; and (d) removing the solvent from the combined extraction solutions obtained in step (c) and drying of the obtained residue.
 2. Method according to claim 1, wherein the extraction in step (a) is carried out once, twice, or three times.
 3. Method according to any one of claims 1 or 2, wherein the solvent in step (a) is selected from the group consisting of n-pentane, n-hexane, and n-heptane.
 4. Method according to claim 3, wherein the solvent in step (a) is n-heptane.
 5. Method according to anyone of claims 1 to 4, wherein the extraction in step (b) is carried out at about 90° C.
 6. Method according to anyone of claims 1 to 5, wherein the solvent in step (c) is 92% (w/w) ethanol.
 7. Method according to claim 1, wherein the solvent in step (a) is n-heptane and the solvent in step (c) is 92% (w/w) ethanol.
 8. Method according to claim 1, wherein the solvent in step (a) is supercritical CO₂ and the solvent in step (c) is 92% (w/w) ethanol.
 9. Extract from hop obtainable according to anyone of claims 1 to 8, characterized by having a content of a bitter acids of at least 0.5%, of xanthohumol of at least 2%, and of prenylated flavones selected from the group comprising 6-prenylnaringenin, 8-prenylnaringenin and isoxanthohumol of at least 0.5%.
 10. Extract from hop according to claim 9, characterized by having a content of a bitter acids of at least 0.8%, of xanthohumol of at least 3%, and of prenylated flavones selected from the group comprising 6-prenylnaringenin, 8-prenylnaringenin, and isoxanthohumol of at least 0.7%.
 11. Pharmaceutical preparation, comprising an extract from hop characterized by having a content of a bitter acids of at least 0.5%, of xanthohumol of at least 2%, and of prenylated flavones selected from the group comprising 6-prenylnaringenin, 8-prenylnaringenin and isoxanthohumol of at least 0.5% and conventional pharmaceutically acceptable additives.
 12. Pharmaceutical preparation according to claim 11, comprising an extract from hop, characterized by having a content of a bitter acids of at least 0.8%, of xanthohumol of at least 3%, and of prenylated flavones selected from the group comprising 6-prenylnaringenin, 8-prenylnaringenin, and isoxanthohumol of at least 0.7%.
 13. Use of an extract from hop as defined in claim 9 or 10 or a pharmaceutical preparation according to claim 11 or 12 for the preparation of a medicament for the prophylaxis and treatment of pathological diseases caused by a deficiency of oestrogens or a dysregulation of sex-hormone-related metabolism, particularly oestrogen metabolism, selected from the group consisting of climacteric complaints, benign prostate hypertrophy, osteoporosis, Alzheimer's disease and diseases of the cardiovascular system.
 14. Use of an extract from hop as defined in claim 9 or 10 or a pharmaceutical preparation according to claim 11 or 12 for the preparation of a medicament for the prophylaxis against sex-hormone-dependent cancers.
 15. Use according to claim 14, wherein the sex-hormone-dependent cancers are selected from the group consisting of breast cancer, carcinoma of the prostate, and carcinoma of the uterus. 